Cellular signal transduction, i.e., the series of events leading from extracellular events to intracellular sequelae, is an aspect of cellular function in both normal and disease states. Numerous proteins that function as signal transducing molecules have been identified, including receptors, docking or recruiting proteins and enzymes such as receptor and non-receptor tyrosine kinases, phosphatases and other molecules with enzymatic or regulatory activities. These molecules generally demonstrate the capacity to associate specifically with either proteins or other signaling molecules (e.g. lipids) to form a signaling complex that can alter cell activity.
Signaling proteins often contain domain(s) of conserved sequence, which serve as non-catalytic modules that direct protein-protein interactions during signal transduction. Such domains include among others, SH2, phosphotyrosine interaction ("PI"), WW and SH3 domains. SH2 and PI domains recognize, i.e., bind to, proteins containing characteristic peptide sequences which include one or more phosphorylated tyrosine residues. WW and SH3 domains recognize proteins containing characteristic peptide sequences which need not contain phosphotyrosine residues. Significant information related to such domains, proteins containing them, the production of proteins containing such domains (including protein fragments and fusion proteins), the characteristic peptide sequences which they recognize and the biological and/or clinical role played by the interactions of such proteins has been described in the scientific literature.
In cases in which the interaction of a particular protein molecule with a binding partner is associated with the cause or symptoms of a disease or pathological condition, compounds capable of interfering with that interaction may be useful in preventing or treating the disease or condition in mammals, including human patients.
Critical tools for the discovery of such inhibitors of protein:protein or other intermolecular interactions are binding assays. The well-known two-hybrid interaction/binding assay described by Song and Fields, Nature, 340:245-247 (1989) has been used to study the interactions of protein-protein interacting partners [See, Fields et al, U.S. Pat. No. 5,283,173 (Feb. 1, 1994)]. Such approaches have also been used to identify presumed SH2 dependent interactions using yeast [Xing, Z. et al., Mol. Biol. Cell, 5:413-421 (1994); and Osborne, M. A. et al., Biotechnol., 13:1474-1478 (1995)] or to detect the inhibition of two-hybrid formation in yeast [Chaudhuri, B. et al., FEBS Lett., 357:221-226 (1995)]. See, also, International patent application No. PCT/US95/03208, incorporated herein by reference for background information on SH3 domains and their ligands including information on the design and preparation of proteins containing various SH3 domains, preparation of peptide ligands for an SH3 domain of interest, and biological/clinical roles of SH3 mediated interactions. See, PCT/US97/02635, incorporated herein by reference, for information on receptor domains (e.g., SH2 and PI domains) for phosphotyrosine-containing ligands, including the design and preparation of proteins containing various SH2 domains, preparation of peptide ligands for an SH2 domain of interest, and biological/clinical roles of SH2-mediated interactions.
Competitive binding assays have been described for detecting test substances which interfere with the association of proteins containing an SH2 domain with their phosphotyrosine containing ligands. See, e.g., Pawson, U.S. Pat. No. 5,352,660. More recently reported binding assays have utilized surface plasmon resonance (Biacore) [see, e.g., Panayotou et al, Mol. Cell. Biol., 13:3567-3576 (1993)] or radioactive ligand based assays. The former has a relatively low throughput, while the latter requires cumbersome filtration manipulations and generates radioactive waste, an increasingly difficult disposal issue.
The availability of materials and methods designed for the rapid and effective identification of inhibitors of protein:protein interactions would be a boon for drug discovery efforts aimed at a wide variety of target protein mediators. It would permit higher-throughput and more efficient identification and development of new pharmaceutical compositions containing inhibitors of intermolecular interactions linked to undesirable or pathological conditions.